Antiserum against 3-0-methyldopamine was produced in rabbits immunized with 3-0-methylhapten conjugated to hemocyanin. The antiserum was used to develop a radioimmunoassay for 3-0-methyldopamine. As little as 0.2 ng/ml of 3-0-methyldopamine can be detected. The major catecholamines and the phenolic aromatic amines and their metabolites did not bind significantly to the antibody. The radioimmunoassay of 3-0-methyldopamine was used to analyze for the endogenous level of 3-0-methyldopamine in urine and plasma of hospitalized children. In children (age 7-157 months), average concentration of 3-0-methyldopamine in urine and plasma was found to be 0.14 micrograms/mg of creatinine (46.25 micrograms/24 hr) and 0.47 ng/ml, respectively. In a child with neuroblastoma, there was a significant increase in the excretion of MD in urine (303 micrograms/24 hr). In adults, the average urine and plasma levels were found to be 95.1 micrograms/24 hr and 0.63 ng/ml.